Selection of quantitative real-time reverse transcription-PCR reference genes in lead-stressed bermudagrass roots treated with different concentrations of melatonin

The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used for gene expression analysis. However, the accuracy of its results depends on the screening of stably expressed reference genes and homogenization of the data. To screen for stably expressed reference genes in the root tip tissue of lead (Pb)-stressed Cynodon dactylon treated with different concentrations of melatonin, the expression levels of 8 candidate reference genes (PP2A, TIP41, CACS, ACT, EF-1α, TUB, UPL7, GAPDH; selected on the basis of preliminary physiological experiments) in 6 samples were determined using qRT-PCR analysis. The results indicated significant differences in the expression levels of the 8 reference genes among the 6 samples, with the Ct values of individual reference genes varying significantly in different samples. The ΔCt, NormFinder, BestKeeper, and geNorm algorithms were used to sort the expression stability of these reference genes. Subsequently, combined with the online tool RefFinder, the comprehensive ranking of the expression stability of each reference gene was calculated. The results revealed that both CACS + PP2A combined was the most suitable reference gene set for the root tip tissue of Pb-stressed bermudagrass treated with different melatonin concentrations. This reference gene combination provides a foundation for analyzing the molecular mechanism through which melatonin influences Pb tolerance, absorption, and transportation in bermudagrass and further promotes the practical application of melatonin in the plant remediation of Pb-contaminated soil.